Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel 38-residue neuropeptide which stimulates adenylate cyclase activity in rat pituitary cells as well as in other neuronal and non-neuronal tissues. In this study we have investigated whether PACAP27 and PACAP38 may stimulate either cyclic AMP accumulation or phosphoinositide formation in cultured cerebellar granule cells. In cultures at 8 days of maturation in vitro (DIV), a 15-min exposure to PACAP27 or PACAP38 equally promoted a concentration-dependent increase in intracellular cAMP content: the effect was significant at 1-5 nM and maximal between 10 and 100 nM, while VIP was 1,000-fold less potent in elevating cAMP levels. In the presence of 3-isobutyl-1-methylxan-thine (200 µM), stimulation by PACAP was present already at 0.1 nM and was maximal (6-fold increase) at 1 nM. A rapid elevation in intracellular cAMP (about 80%) was observed within a 30-second exposure to 10 µM PACAP38 or PACAP27; the maximal activity of PACAP was present between 15 and 30 min and progressively declined at 60 min without reaching basal values. PACAP27 and PACAP38, but not VIP, were also able to stimulate inositol phospholipid hydrolysis: PACAP38 (EC50: 0.16 nM) was 10-fold more potent than PACAP27 (EC50: 2.1 nM) in stimulating [3H]inositol phosphate formation. The effect of PACAP was rapid: fractionation of [3H] inositol phosphates revealed that inositol trisphosphate and inositol bisphosphate increased earlier (within 20 s) than inositol monophosphate (within 60 s). Stimulation of inositol phospholipid hydrolysis by PACAP was not mediated through the activation of the adenylate cyclase-cAMP system because neither 10 µM forskolin nor VIP (1–10 µM) were able to affect inositol phospholipid turnover. Interestingly, while the cAMP response was already present (and maximal) at 2 DIV, PACAP increased phosphoinositide hydrolysis only at 4 DIV. Our results provide clear evidence for the presence of PACAP receptors linked to both the adenylate cyclase-cAMP system and the phosphoinositide turnover in cerebellar granule cells.

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